Carbonic anhydrase (CA) is a family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. Out of fifteen CA isoforms (CA I, II, III, VA, VB, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, and XV) present in human, twelve of them display catalytic activity. Three isoforms CA VIII, X and XI are non-catalytic and are termed as CA related proteins. Apart from the differences in catalytic efficiency, 12 active isoforms also differ in cellular, localization, tissue distribution, and involvement in physiological processes. Furthermore, aberrant expression of the enzymes is commonly associated with a host of diseases. These include: glaucoma (CA II, IV), cancer (CA IX, XII), edema (CA II), sterility (CA XIII), altitude sickness (CA II), obesity (CA VA) and hemolytic anemia (CA I). Out of 15 CA isoforms (alpha-class CAs), CA IV, IX, XII, XIV isoforms are associated with cell membrane. While both CA IX and XII express in solid tumors, CA IX has been shown to express more prevalent in solid tumors and exhibiting low expressions in normal tissues thereby making it an excellent biomarker for targeted-drug deliver for cancers.
The CA9 gene encodes for a 459 amino acid transmembrane glycoprotein that exists as a homodimer. It is comprised of: a proteoglycan-like domain (PG) (59 aa), catalytic domain (CA) (257 aa), a signal peptide domain (which is removed prior to enzyme maturation) (37 aa), transmembrane domain (TM) (20 aa), and a C-terminal intracellular domain (25 aa). Mass spectroscopy and X-ray crystallography have confirmed the presence of an intermolecular disulfide bridge between adjacent Cys137 residues of the mature homodimer that, coupled with a region of hydrophobic residues, are proposed to stabilize the dimer interface. N-linked and O-linked glycosylation sites also exist at Asn 309 and Thr 78, respectively.
The catalytic domain of CA IX is structurally homologous to the alpha-CAs with high amino acid conservation within the active site. The active site is located in a larger conical cavity (15 Å deep), which spans from the surface to the center of the protein. The zinc atom is located at the bottom of the cavity. In CA IX three histidine residues (His 226, 228 and 251, as numbered in the full length aa sequence) coordinate the zinc ion at the base of the active site cleft; in the crystal structure (PDB ID: 3IAI) sulfonamide amine group in the acetazolamide (AZM) displaces a zinc bound water/hydroxide (Zn—OH/H2O) molecule maintaining a tetrahedral coordination about the zinc ion. Variability between the CA isoforms occurs in the hydrophobic and hydrophilic pockets of the active site and surface amino acids. In CA IX, Leu-223, Val-253, Val-263, Leu-267, Leu-273, Leu-330, and Pro-334 define the hydrophobic region, while Asn-194, His-196, Ser-197, Gln-199, Thr-201, and Gln-224 identify the hydrophilic one.
The catalytic efficiency of CA IX is fast and comparable to that of CA II; CA II exhibits a kcat of 1.4×106 while CA IX has a kcat of 3.8×105. The presence of the PG domain in CA IX is unique compared to the other CA isoforms and is thought to be responsible for its cell adhesion capability and maintaining its catalytic activity in the acidic tumor microenvironment.
The most critical role of CA IX is thought to be extracellular pH regulation, especially in the tumor microenvironment. Proliferating cancer cells often produce large amounts of lactate, carbon dioxide and protons during oncogenic metabolism making CA function pivotal in tumor cell survival. These metabolic products accumulate in the extracellular environment and significantly lower the extracellular pH. In order to maintain a near physiological intracellular pH, bicarbonate anions generated by CA IX during the hydrolysis of carbon dioxide are transported into the cell via anion transporters to buffer intracellular pH levels. In addition protons produced from the reaction remain extracellular thus contributing to the acidic nature of the tumor milieu. Disruption of this regulatory pathway would therefore have detrimental effects on overall tumor cell survival.
In a non-disease state CA IX expression is limited to the gut epithelium; specifically, the basolateral surfaces of the cryptic enterocytes of the duodenum, jejunum and ileum. The most prominent levels of CA IX are seen in these proliferating crypt cells suggesting that CA IX may be involved in intestinal stem cell proliferation and regulation of certain metabolic functions. Northern blot and immunohistochemical staining have also confirmed that CA IX expression in the ovarian coelomic epithelium, cells of hair follicles, pancreatic ductal cells and fetal rete testis. In addition high levels of CA IX are observed in developing embryonic tissues of the gut, lung and skeletal muscle and decrease in adult tissues. These observations indicate CA IX expression is primarily associated with areas of low pH and high rates of cell proliferation in normal tissues. Whether or not this makes CA IX a regulatory element in normal tissues has not been confirmed.
CA IX is ectopically expressed in a variety of neoplastic tissues. Expression has been observed in malignancies of the breast, lung, kidney, colon/rectum, cervix uteri, oral cavity, head/neck, gallbladder, liver, brain (high-grade), pancreas, and gastric epithelium. No differences exist between the cDNA of CA IX isolated from normal and tumor tissues, which implies similar physiological function in both tissues. CA IX expression depends on Hypoxia-inducible factor 1 (HIF-1) activation [via the upregulation of HIF-1α or the down regulation of Von Hippel-Linadau (VHL)]. Specifically, the activation of the HIF-1 mediated pathway that induces CA IX expression can be due to a reduction in cellular O2 levels, an activation of signaling pathways via the presents of growth factors and inflammatory response elements, and in some cases due to mutations in the tumor suppressor, VHL as seen in cases of renal cell carcinoma (RCC) where CA IX is homogenously expressed. More recently, CA IX has shown to have significant expression levels in stromal cells that are engaged in a molecular cross-talk circuitry with cancer cells. Specifically, CA IX has been shown to be expressed in cancer-associated fibroblasts (CAFs) via redox-based stabilization of HIF-1. It is postulated that expression of CA IX in CAFs provides the acidic extracellular environment necessary to drive epithelial-mesenchymal transitions (EMTs) in adjacent cancer cells. Summation of these findings indicates CA IX as a diagnostic marker of events of tumor hypoxia in many solid tumors.
CA IX expression levels also serve as prognostic markers for several cancer types. Specifically, patients suffering from brain, breast, cervical, rectal or lung cancer that also display high levels of CA IX typically show a poorer prognosis. In contrast, for clear cell renal cell carcinoma patients low CA IX levels indicate poor clinical outcome. CA IX's contribution to maintaining the hypoxic tumor microenvironment is highly correlated to patient prognosis thus making it both a biomarker and drug target.
Hypoxia is a condition commonly seen in metastatic tumors where cells are deprived of oxygen due to rapid proliferation and a shift in their metabolism. Specifically, hypoxic tumor cells outgrow their blood supply leading to regions of low oxygen concentration (typically ≤1% of overall oxygen content) as well as a decrease in extracellular pH (˜pH 6.5) in the tumor microenvironment. This hypoxic stress induces a shift in the tumor cells general metabolism from oxidative phosphorylation in the mitochondria to aerobic glycolysis in the cytosol as the main energy source. Interestingly, this metabolic shift remains present in the tumor cells regardless of the amount of the available O2 in the given environment; a phenomenon often described as the Warburg effect. Since these tumor cells rapidly use glycolysis, increased amounts of lactic acid are exported from the cell, thus lowering the extracellular pH. As a result, there is an upregulation of pH homeostasis factors in tumor cells to establish a regulated intracellular/extracellular pH gradient.
Since the 1930s it has been well established that there is a correlation between tumor hypoxia and a resistance to radiation therapy. In addition, hypoxic tumors have shown to also present a resistance to common chemotherapeutics and a high probability of metastases; hence tumor hypoxia has been associated with a poor patient prognosis. Hypoxia inducible factors (HIF) are key regulators of the hypoxic-induced stress response in both normal and tumor cells. Specifically, increased HIF-1 has been associated with activating hypoxia-inducible genes that express hypoxia-responsive elements (HRE) that upregulate elements associated with metabolism, cell proliferation, drug resistance, pH regulation, angiogenesis, metastasis, and the overall progression of cancer. In order to survive in the acidic microenvironment these tumor cells must be able to maintain an intracellular pH at or near physiological levels (pH 7.4). Therefore CA activity is key in this regulatory process.
CA IX expression directly correlates to an upregulation of HIF elements, and has been shown to play a role in tumor cell survival, proliferation, migration, growth, adhesion, pH regulation, and cell-signaling pathways. The minimal expression of CA IX in normal tissues and its location on the external interface of tumor cells have made it an attractive therapeutic target. As a result, several methods have been employed to try to target CA IX in terms of isoform selective small-molecule inhibition, location specific targeting, knockdown using RNAi technology, and more recently antigenic targeting of CA IX as a means to deliver anti-cancer therapeutics directly to tumor.
CAIs have been extensively studied and their inhibition mechanisms are well established. Sulfonamide-based compounds are the most potent and most utilized among the CAI classes. These compounds bind to the zinc ion via a sulfonamide as the zinc-binding group (ZBG) in a deprotonated form displacing the zinc bound water/hydroxide molecule while still maintaining the tetrahedral coordination about the zinc ion. Though some sulfonamides display inhibition constants in the sub-nanomolar range for CA IX, they also inhibit other isoforms of CA. This is due to the conserved architecture of the active site among the human CAs. For all the catalytic human CAs, the three histidines coordinating the zinc, Thr 199 (CA II numbering; termed the “gatekeeper”), and Glu 106 are conserved. Both T199 and E106 play roles in catalysis. T199 hydrogen bonds to the zinc bound water/hydroxide via its OH group, while E106 hydrogen bonds to T199.
Small molecular weight CA inhibitors (CAIs) that utilize a ZBG tend to bind deep into the active site cavity and do not make extensive interactions with amino acids that vary between the CA isoforms, thus contributing to their indiscriminatory inhibition profiles. As a result, alternative approaches have been developed for better isoform specific CAIs, with the “tail-approach” being one of the most successful. In the “tail approach” a chemical moiety (known as the tail) is appended onto an organic scaffold of a ZBG (for example heterocyclic or aromatic). This tail elongates the inhibitor allowing it to make extensive interactions with amino acids towards the outside of the active site. The addition of these tails can also alter the properties of the CAI, for example making it more soluble by the addition of a tail that is hydrophilic in nature, or manipulating the overall charge of the compound; such as cationic CAIs. The use of structure-based drug design has proven a valuable technique to exploit the subtle differences existing between the active site of the various isoforms. For example, utilizing steroidal based sulfonamides as lead compounds has led to the development of several similar CAIs that are able to exploit CA IX's larger hydrophobic pocket by increasing the number of hydrophobic interactions via van der Waals contacts.
Despite the promise of structural exploitation of the CA IX active site to improve upon current and novel CAIs, the expression and crystallization of wild type CA IX has been an arduous challenge and thus made it difficult to carry out extensive structural analysis. A CA IX-mimic has been engineered, it is a modified CA II (an enzyme that is routinely expressed and crystallized) that contains active site mutations specific to CA IX. This has provided a useful template to rapidly analyze and predict modes of binding of CAIs to CA IX. Structural analysis of several CAIs has made it possible to design drugs that exhibit both location specific targeting and prodrug like properties that have shown to be useful in selectively inhibiting CA IX.
Apart from the development of small-molecule inhibitors, CA IX specific antibodies and their conjugates have also been engineered with some are currently in Phase III clinical trials (RECENARX). M75 and G250 are two such monoclonal antibodies that recognize the enzymes proteoglycan domain. Upon binding to CA IX these antibodies cause a reduction in tumor cell adhesion and motility, and induce natural killer cells to target tumor cells for eradication. The development of monoclonal antibodies with high binding affinity eliminates the problem of off-target effects commonly encountered in CAI drug design.
The extracellular location of the active site of CA IX presents an alternative method of targeting the enzyme in tumor cells. Specifically, CAIs can be designed that have physiochemical properties that allow them to be impermeable to the plasma membrane; hence decreasing the chance of inhibiting off-target cytosolic CAs observed by classic CAIs. This presents a drug design strategy that incorporates location specific targeting of CA IX rather than exploiting differences in inhibition profiles alone. To date several compounds that show limited membrane permeability have been synthesized and designed. Such compounds utilize bulky chemical moieties, such as in albumin-acetazolamide, or exploit charged moieties in the form of fluorescently labeled sulfonamides or cationic sulfonamide derivatives. The design of such CAIs employs essentially two distinct rationales: (1) high molecular weight compounds that are simply too bulky to cross the plasma membrane, or (2) a cationic moiety that is incapable of permeating into the reduced cytosolic environment. Despite both types of compounds showing favorable inhibition and membrane impermeability, the use of cationic sulfonamides has shown to be the more feasible option for drug development since high molecular weight compounds often induces potent allergic reactions and reduced bioavailability in vivo. As a result several cationic sulfonamides have been developed using quaternary ammonium sulfate (QAS) as a lead compound, or fluorescently labeled sulfonamide derivatives.
Glycoconjugated sulfonamides, a more recent class of CAIs, have shown to exhibit both membrane impermeability and isoform selective inhibition of CA IX. These particular CAIs utilize benzene sulfonamides, sulfonamides, or cyclic secondary sulfonamides conjugated to a mono- or disaccharide tail. The design of these CAIs was through the influence of the clinically used Topiramate (anti-epileptic therapeutic). Most likely the reason these compounds do not permeate into the cell is due to their high molecular weights, and the addition of a sugar moiety that is not easily transported. Furthermore, unlike previously used bulky sulfonamide derivatives, the addition of a sugar moiety allows these CAIs to maintain water-solubility, and thus maintain good bioavailability. Another promising aspect is that these CAIs show an impressive inhibition profile, with a >1000-fold selectivity for CA IX over CA II in some cases. Also, the carbohydrate attachment presents an area of manipulation on these CAIs where cleavable ester bonds can be added to the carbonyls of the carbohydrate tail allowing the CAI to be “packaged” in the form of a prodrug. Although these compounds present great promise in terms of developing a drug for CA IX, the use of carbohydrate moieties poses a potential dilemma. That is, the use of a carbohydrate, specifically a monosaccharide, might unintentionally interact with glucose transporters, similar to statins, in which myotoxicity was observed. However, this notion has not been tested. Interestingly, a way to circumvent such an issue would be the development of sucrose-based conjugates that would have no interactions with specific transporters due to the lack of sucrose transporters in human tissue. Interestingly, the current disaccharide-conjugates that have been developed into CAIs utilize a galactose moiety and show stronger inhibition for CA II versus CA IX. Although these compounds will not enter the cytosol, they may not bind to CA IX tightly enough to be considered a valid drug candidate. However, utilization of other disaccharide-based compounds, such as the suggested sucrose-conjugate mentioned previously, might show higher inhibition for CA IX, and thus present a CAI that is selective for CA IX in both location specificity and direct inhibition.
There are many publications containing both CA IX targets and NIR dyes. One work targets CA IX by synthesizing sulfonamide derivatives and testing them both in vitro and in vivo [Kevin Groves, Bagna Bao, Jun Zhang, Emma Handy, Paul Kennedy, Garry Cuneo, Claudiu T. Supuran, Wael Yared, Jeffrey D. Peterson, Milind Rajopadhye, Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors, Bioorganic & Medicinal Chemistry Letters, Volume 22, Issue 1, 1 Jan. 2012, Pages 653-657, ISSN 0960-894X, http://dx.doi.org/10.1016/j.bmcl.2011.10.058.]. Groves et al. synthesized sulfonamide derivatives and used amide linkage to couple them to succinimidyl esters of one or more of four commercially available hydropobic indocyanine NIR fluorochromes. They show localization of the synthesized sulfonamide derivatives to tumors in HT-29 tumor bearing mice.
A CA IX targeted agent is validated by Bao et al. for in vivo detection of CA IX expressing tumors [Bao B, Groves K, Zhang J, Handy E, Kennedy P, Cuneo G, et al. (2012) In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia. PLoS ONE 7(11): e50860. doi:10.1371/journal.pone.0050860]. Localization was compared using a CA IX antibody.
Groves and Bao are both inventors on U.S. Patent Application Publication 2012/0321563, which discloses imaging agents that target carbonic anhydrase. The '563 claims a carbonic anhydrase targeting agent composed of a sulfonamide carbonic anhydrase binding moiety (CAB) that is linked to a linker L, and then to an optional Q group, and then finally to a NIR chromophore. The claimed NIR chromophore is a genus structure for the closed chain subgroup of the cyanine dye family.
Claudiu Trandafir Supuran is a co-inventor of International Patent Publication No. WO 2014/136076 titled “Assembly comprising an absorber of near infrared (NIR) light covalently linked to an inhibitor of carbonic anhydrase”. The absorber of NIR light has an optical absorption cross section not lower than 100 nm2.
Neri and co-workers use small molecule drug conjugates to target CA IX expressed in solid tumors in vivo [Krall, N., Pretto, F., Decurtins, W., Bernardes, G. J. L., Supuran, C. T. and Neri, D. (2014), A Small-Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors. Angew. Chem. Int. Ed., 53: 4231-4235. doi:10.1002/anie.201310709]. They prepare CAIX ligand-linker-dye conjugates, and show that it preferentially accumulates in subcutaneous CAIX-expressing SKRC52 tumors in nude mice. Claudiu Supuran is also an inventor in U.S. Pat. No. 8,628,771 B2 which discloses methods for inhibiting growth of cells that express CA IX, methods to screen for CA IX specific inhibitors, methods to visualize and image tissues that selectively bind the activated CA IX, methods to target cells that have expressed CA IX, and methods utilizing CA IX specific inhibitors coupled to gene therapy agents. The '771 uses a CA IX-specific antibody conjugated to a radioisotope to target and detect CA a.
In another work, Neri and co-workers show that a bivalent ligand against the tumour marker carbonic anhydrase IX leads to an improved tumor targeting performance compared with the corresponding monovalent counterpart in the SKRC52 model of constitutively CAIX-positive renal cell carcinoma [Krall, Nikolaus, Francesca Pretto, and Dario Neri. “A bivalent small molecule-drug conjugate directed against carbonic anhydrase IX can elicit complete tumour regression in mice.” Chem. Sci. 5.9 (2014): 3640-3644.]. The acetoazolamide derivatives are linked to monovalent and bivalent dye conjugates utilizing the commercially available dye, IRDye750.
Pomper and co-workers report on the synthesis and in vivo performance of [111In]XYIMSR-0, a modified dual motif CAIX inhibitor for nuclear imaging of the clear cell subtype of renal cell carcinoma inspired by the earlier bivalent work by Neri et al. [Yang, X., et al. “Imaging of carbonic anhydrase IX with an 111In-labeled dual-motif inhibitor.” Oncotarget 6.32 (2015): 33733-33742.]. Pomper replaced the IRDye750 portion of the molecule with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a more hydrophilic species that also enables convenient radiolabeling with metal isotopes for positron emission tomography, single photon emission computed tomography, and radiopharmaceutical therapy. Indium-111 is used as the radionuclide for its relatively long half-life (2.8 day) to enable extended monitoring of pharmacokinetics. A FITC label is used as standard to measure CAIX binding affinities in the radiotracers.
In another work, Neri and co-workers describe the synthesis of an acetazolamide-based carbonic anhydrase ligand with high affinity for the tumor associated isoform CAIX, labeled with 99m Tc, a widely-used gamma-emitting radionuclide for nuclear medicine applications [Nikolaus Krall, Francesca Pretto, Martin Mattarella, Cristina Müller, and Dario Neri, A technetium 99m-labeled ligand of carbonic anhydrase IX selectively targets renal cell carcinoma in vivo, J Nucl Med jnumed.115.170514 published ahead of print Feb. 18, 2016 (10.2967/jnumed.115.170514).].
Supuran and co-workers describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate “tail” moiety [Moeker, Janina, et al. “Structural insights into carbonic anhydrase IX isoform specificity of carbohydrate-based sulfamates.” Journal of medicinal chemistry 57.20 (2014): 8635-8645.]. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. Their structural analysis indicates that there exist two distinct modes of binding between CA IX and CA II of compound 5e of Moeker et al., however, in both cases, this compound interacts with the hydrophilic pocket of the enzyme. As this pocket is generally conserved between CA II and CA IX, it may account for the nanomolar binding affinities between both enzymes. In contrast, compound 5d of Moeker et al., which showed a differential inhibition profile between CA II and CA IX, binds to the CA IX active via interactions with the hydrophobic pocket. This region in the CA active site contains more variability between residues of CA II versus CA IX. As a result, this region has been termed as one of the “selective pockets” in the CA active site.
The X-ray structure of the catalytic domain of CA IX shows a fold that is significantly different from the other CA isoforms in quarternary structure [Alterio, Vincenzo, et al. “Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX.” Proceedings of the National Academy of Sciences 106.38 (2009): 16233-16238.]. They conclude that the region 125-137 differs both in structure and sequence in all these isozymes, and it represents a “hot spot” to be considered in structure-based drug design.
Treating cancer typically requires the use of several therapeutic strategies such as surgery, radiation therapy, and/or chemotherapies. Often therapies must be combined due to efficacy of one preceding the other. For example surgery and radiation therapy, although effective in a vast majority of cases, present limitations in that they can only target confined local regions of neoplastic tissue and are not effective at treating highly metastatic cancer cases. At this stage combinations of multiple chemotherapeutics are usually employed in an attempt to kill cancer cells that have migrated from the primary tumor site. Furthermore, highly aggressive and hypoxic tumors often develop resistance to radiation and certain chemotherapies, or are inoperable; hence alternative or combinations of chemotherapeutics are the only method of treatment available in these particular cases. This feature of hypoxia and its association with resistance to radiation and chemotherapies has been observed in several cancer types. This is most likely due to several factors including a reduction in overall O2 content making the generation of free-radicals needed for radiation therapy extremely difficult, the reduced extracellular pH disrupting functions of alkylating agents, and an upregulation of drug-resistance factors induced by HIFs. CA IX, has been linked to cases of therapeutic resistance for several cancers, and is often used as a biomarker for radiation resistance. As such evidence suggests labeling CA IX via a active site binding moiety linked to a NIR dye allows localization of hypoxic cancer cells, indicating its potential use as a means to assist surgeons in removing cancerous tissue during surgery.
Ferreira and co-workers compared the in vitro cytotoxicity of four NIR Dyes: IR125, IR780, IR813, and IR820 [Conceição, David S., Diana P. Ferreira, and Luis F. Vieira Ferreira. “Photochemistry and Cytotoxicity Evaluation of Heptamethinecyanine Near Infrared (NIR) Dyes.” International journal of molecular sciences 14.9 (2013): 18557-18571.]. One source of cytotoxicity is due to after the intersystem crossing to the triplet state, the sensitizer can interact with molecular oxygen via a triplet-triplet annihilation process, generating singlet oxygen, or, alternatively, the sensitizer in its triplet state can participate in electron transfer processes or radical intermediate formation, also leading to the generation of reactive oxygen species. But Ferreira proposed that the main cause for the significant values of cytotoxicity presented by IR125 and IR813 should be related with their instability in solution (during long periods of time), and degradation products and photoproducts that arose during the inoculation of the dyes in the cellular culture. The addition of a cyclohexenyl ring promoted a significant molecular stabilization, and IR820 is the only NIR dye examined that exhibited no major cytotoxic effects, both in the presence and absence of light.